The goal of this project is to characterize the pathway of synthesis and distribution of Alpha-granule (fibrinogen, Beta-thromboglobulin and platelet factor 4) and plasma membrane (GPIIb-IIIa) proteins by megakaryocytes. These proteins will be identified in human and baboon megakaryocytes from bone marrow aspirates using electron microscopic and immunocytochemical techniques previously developed by the investigators to study these substances in platelets. Well-characterized mono and polyclonal antibodies will be used to localize the antigens in subcellular organelles: presence of antigens in the rough endoplasmic reticulum and Golgi complex will be used as evidence of their synthesis by megakaryocytes. The stage of maturation at which each of these substances is synthesized as well as the mechanisms by which they are distributed into cellular organelles will be determined. Identification of antigens in endocytotic organelles (coated vesicles, multivesicular bodies or lysosomes) alone, would indicate the presence of these substances in the cell is due to internalization. Incubation of megakaryocytes with (3H) antigens followed by autoradiographic localization will provide additional evidence for endocytosis. We also plan to characterize thrombin binding and internalization by megakaryocytes as well as the effects of thrombin on cellular organelles such as Alpha-granules and their contents. In order to facilitate these studies immune-affinity techniques for isolating megakaryocytes will be developed using anti-GPIIb-IIIa antibodies in conjunction with established methods. Additional monoclonal antibodies against cell surface antigens will be characterized and used to extend our studies of the megakaryocyte plasma membrane. These experiments may also provide new information on formation of the demarcation membrane during megakaryocyte maturation. In addition, synthesis and distribution of Alpha-granules and plasma membrane proteins will be studied in conditions in which these cellular functions may be altered. These processes will be studied in megakaryocytes obtained from 1) baboons made thrombocytopenic with anti-platelet antisera as well as humans with consumptive platelet disorders to determine the effects of stimulation of megakaryocytopoiesis, and 2) patients with hereditary platelet disorders in which there is deficiency of substances found in plasma membranes (Glanzmann's thrombasthenia) or Alpha-granules (Gray platelet syndrome). These studies may provide information which will advance our understanding of the pathophysiology of these diseases.